The grant application deals with the characterization of estrogen (E) target cells in long-term culture. These cells, established in our laboratory, are derived from rat pituitary tumor and from normal 10-day-old rat endometrial cells. They have estrogen receptor of high affinity, low capacity and are saturable. These cells grow as tumors at the site of inoculation in inbred rats. The objectives to be pursued include: 1) The isolation of variant clones that would carry receptor differing in location, amount and properties intrinsic to the binding to estrogens. 2) The inter- and intraspecific somatic fusion of these E target cells with other cells will be done. These somatic cell hybridization experiments are designed to explore the control under which the E target cell expresses its ability of binding specifically to E, and its malignant properties. 3) The conditions under which E target cells demonstrate their dependency on E will be defined. We will compare growth conditions using serum-supplemented media from fetal, newborn, castrated and adult female rats as well as enriched fractions from rat plasma proteins. Methods to be used include cell culture, somatic cell hybridization, chromosome banding techniques, determination of specific binding (separation of free and bound steroids) by charcoal-dextran adsorption, hydroxyapatite adsorption, protamine sulfate precipitation, density gradient centrifugation. The development of the above mentioned topics will help to understand the mechanism of action of estrogens in normal and malignant E target cells.